sonogenetics for noninvasive and cellular-level ... · sonogenetics, which uses ultrasound to...

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Sonogenetics for noninvasive and cellular-level neuromodulation in rodent brain

Yaoheng Yang1, Christopher Pham Pacia1, Dezhuang Ye2, Lifei Zhu1, Hongchae Baek1, Yimei

Yue1, Jinyun Yuan1, Mark J. Miller3, Jianmin Cui1, Joseph P. Culver1,4,5, Michael R. Bruchas6,

and Hong Chen1,7*

1. Department of Biomedical Engineering, Washington University in St. Louis, Saint Louis, MO

63130, USA.

2. Department of Mechanical Engineering and Materials Science, Washington University in St.

Louis, Saint Louis, MO 63130, USA

3. Division of Infectious Diseases, Department of Medicine, Washington University School of

Medicine, Saint Louis, MO, USA

4. Mallinckrodt Institute of Radiology, Washington University School of Medicine, Saint Louis,

MO 63110, USA.

5. Department of Physics, Washington University in St. Louis, Saint Louis, MO 63110, USA

6. Department of Anesthesiology and Pain Medicine. Center for Neurobiology of Addiction,

Pain, and Emotion. University of Washington, Seattle, WA 98195, USA

7. Department of Radiation Oncology, Washington University School of Medicine, Saint Louis,

MO 63108, USA.

*Address correspondence to Hong Chen, Ph.D. Department of Biomedical Engineering and

Radiation Oncology, Washington University in St. Louis. 4511 Forest Park Ave. St. Louis, MO,

63108, USA. Email: [email protected]

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 28, 2020. . https://doi.org/10.1101/2020.01.28.919910doi: bioRxiv preprint

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Abstract

Sonogenetics, which uses ultrasound to noninvasively control cells genetically modified with

ultrasound-sensitive ion channels, can be a powerful tool for investigating intact brain circuits.

Here we show that TRPV1 is an ultrasound-sensitive ion channel that can modify the activity of

TRPV1-expressing cells in vitro when exposing to focused ultrasound. We also show that

focused ultrasound exposure at the mouse brain in vivo can selectively activate neurons that are

genetically modified to express TRPV1. We demonstrate that precise manipulation of neural

activity via TRPV1-based sonogenetics can be achieved by spatiotemporal control of ultrasound-

induced heating. The focused ultrasound exposure is safe based on our inspection of neuronal

integrity, apoptosis, and inflammation markers. This sonogenetic tool enables noninvasive, cell-

type specific, spatiotemporally controlled modulation of mammalian brain activity.


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Introduction

Technologies for noninvasive modulation of mammalian brain activity with

spatiotemporal precision and cell-type specificity have long been desired for experimental

investigation of intact neural systems. Compared with electric, magnetic, optical, and chemical

stimuli that have been used by existing neuromodulation technologies, focused ultrasound (FUS)

offers unique advantages as it can noninvasively deliver ultrasound energy through the intact

human scalp and skull deep into the brain and focus at cortical areas1,2 as well as deep brain

areas3 with high spatial selectivity. Existing FUS neuromodulation techniques use ultrasound

pulses with low intensity to produce mechanical effects for neural and behavioral modulation

with a negligible temperature increase4. Numerous studies have confirmed that FUS can

noninvasively modulate neural activity and brain function in animal models5,6 and humans1,3.

However, the broad application of FUS neuromodulation is challenged by the wide variation in

neural cell types in the sonicated brain tissue, resulting in neuromodulation with relatively low

reliability and replicability7,8. Advances in genetics-based tools enable manipulation of a specific

type of neurons embedded within densely-wired brain circuits for assessing the causal role that

different groups of neurons have in controlling circuit activity and behavior outcomes. Among

genetics-based tools, optogenetics has been transforming neuroscience research by allowing

targeted control of precisely defined events in the brain via light stimulation; however, it requires

invasive surgery to permanently implant devices for delivering light deep into the brain.

Similar to optogenetics, sonogenetics intends to use ultrasound to precisely control the

activity of neurons that have been genetically modified to express ultrasound-sensitive ion

channels (i.e., sonogenetics actuators). The major challenge in the development of sonogenetics

is to find suitable actuators. Sonogenetics was initially proposed by Ibsen et al9 where ultrasound


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selectively activated the cells expressing a mechanosensitive ion channel (TRP-4) in

Caenorhabditis elegans. However, ultrasound alone did not affect the behavior of the worm.

Behavioral changes were observed only when the body of the worm was surrounded by

microbubbles added to the agar plate where the worm was placed. The requirement for adding

microbubbles to activate the mechanosensitive signaling prevents the translation of this method

to studies in the mammalian brain. A recent study using Caenorhabditis elegans found that the

mechanosensitive ion channel MEC-4 was important for ultrasound neuromodulation in the

absence of microbubbles10. But, its application in mammalian brains is limited because high-

frequency ultrasound (10 MHz) used in that study cannot efficiently penetrate through the skull.

Other mechanosensitive ion channels, for example, TREK-111, MscL12, Piezo113,14, and TRPA115

have been proposed as potential sonogenetic tools based on in vitro cell culture studies without

in vivo validation. A most recent study showed that an engineered auditory-sensing protein

(prestin) caused neurons in the mouse brain to sense ultrasound stimulation16; however, this

protein, which is not an ion channel, lacks the ability to control neuron activity in a temporally

precise fashion. To our knowledge, there is so far no study that has identified ultrasound-

sensitive ion channels for spatiotemporally precise modulation of neural activity in mammalian

brains.

Ultrasound propagation in tissue generates not only mechanical effects but also thermal

effects owing to viscous dissipation as molecules move back and forth in the ultrasound field.

Here, we present a sonogenetic tool (Fig. 1) for noninvasive, spatiotemporally controlled

manipulation of cells genetically modified to express the thermosensitive ion channel – transient

receptor potential vanilloid 1 (TRPV1). The activation temperature of TRPV1 is around 42 °C17,

which is only a few degrees higher than the body temperature to permit quick and safe


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stimulation while allowing the channels to be closed at physiological temperature and

minimizing other potential thermal effects on the neural circuits18,19,20. This technique opens new

avenues for noninvasive, cell-type specific, spatiotemporally precise control of mammalian

neural activity.

Fig. 1. Schematic illustration of TRPV1-mediated sonogenetics. FUS directly and selectively

activates neurons expressing the thermosensitive ion channel TRPV1 by spatiotemporally

controlling the heating of targeted brain tissue to ~42 °C.

Results

TRPV1 is a sonogenetic actuator

Our first experiment was performed to determine whether TRPV1 is a sonogenetic

actuator by evaluating whether ultrasound could selectively control intracellular Ca2+ influx in

TRPV1-expressing HEK293T cells. We developed an experimental setup that allows

simultaneous fluorescence imaging and FUS stimulation of HEK293T cells. This setup used a


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ring-shape FUS transducer that was placed underneath the cell culture plate, allowing

fluorescence imaging through the center opening of the ring (Fig. S1). Temperature rise

associated with the ultrasound exposure was measured using a fiber-optic thermometer placed ~1

mm away from the center point of the microscope’s field of view (FOV). The maximum

temperature induced by FUS as recorded by the thermometer was 42.0 ± 0.2 °C. The

functionality of TRPV1 was confirmed by the observation of Ca2+ influx in response to capsaicin,

a TRPV1 agonist (Fig. S2).

A high-amplitude Ca2+ influx was detected in the FUS-stimulated TRPV1-expressing

cells (TRPV1+) using fluorescence imaging (Fig. 2a, 2c). The maximum increase in the

fluorescence intensity(ΔF/F) was 0.46 on average (Fig. 2e). The TRPV1 antagonist

Capsazepine21,22 dramatically reduced Ca2+ influx in response to FUS stimulation in TRPV1+

cells, suggesting that the observed Ca2+ influx was due to TRPV1 activation (Fig. S3). To

determine whether FUS stimulation affected cells not expressing TRPV1, we stimulated cells

that did not express TRPV1 (TRPV1-) and did not evoke significant Ca2+ influx (Fig. 2c, 2d). In

both TRPV1- and TRPV1+ cells, there were no significant changes in intracellular Ca2+

concentration without FUS stimulation. FUS stimulation had no detectable effects on cell

viability (Fig. S4). These findings suggest that TRPV1 can serve as a sonogenetic actuator.

To test our hypothesis that FUS activates the TRPV1+ cells through FUS thermal-

dominated effects, we performed an experiment to heat the TRPV1+ cells by water bath heating.

We controlled the heating duration to increase the temperature to 42.2 ± 0.5 °C, which was

approximately the same level as FUS-induced heating. We found that water-bath heating on

TRPV1+ cells had a similar success rate and mean fluorescence intensity as FUS stimulation (Fig.

2). While we cannot rule out the contribution by the ultrasound mechanical effect, these findings


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suggest that the activation of the TRPV1 ion channel is dominated by the ultrasound thermal

effect.

Fig. 2. TRPV1 enables FUS activation of HEK293T cells in vitro. (a, b) Fluorescence images

of HEK293T cells. Red: HEK293T cells expressing mCherry with TRPV1 ion channel (TRPV1+,

top row) or without TRPV1 ion channel (TRPV1-, bottom row). Green: Intracellular Ca2+ before

and after (a) FUS stimulation and (b) water-bath heating. (c) Heatmap of normalized Ca2+

fluorescence intensity change (ΔF/F) of 100 randomly selected TRPV1+ and TRPV1- cells from


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3 independent trials in the control group without any stimulation (left panel), FUS group (middle

panel), and heating group (right panel), respectively. (d) Percentage of TRPV1+ and TRPV1-

cells within the field of view of the fluorescence microscope that responded to FUS stimulation

and water-bath heating, respectively. Cells in the control group were not stimulated. The error

bar indicates the standard error of the mean (SEM). Normalized fluorescence intensity change

(ΔF/F) of TRPV1+ and TRPV1- induced by (e) FUS stimulation and (f) water bath heating. The

solid lines indicate the mean and shaded gray represents SEM. The insert shows the

corresponding temperature curve.

Sonogenetics in the mouse brain

After establishing TRPV1 as a sonogenetic actuator through the in vitro experiment, we

then tested whether FUS could directly activate TRPV1+ neurons in the mouse brain in vivo. We

used a two-photon microscope (2PM) combined with genetically encoded calcium indicator

(GCaMP6f) for in vivo neural activity recording in the mouse cortex. Calcium imaging allows

direct observation of brain activity and overlaying functional activity with genetic and

anatomical identity23. We intentionally avoided using electrophysiological recording because

electrodes inserted in the brain interfere with ultrasound wave propagation, and ultrasound wave-

induced mechanical vibration generates artifacts in the electrical recording.

The TRPV1 transgene was placed under the excitatory neuronal promotor calmodulin

kinase II α-subunit and before mCherry by posttranscriptional cleavage linker p2A (CaMKII-

TRPV1-p2A-mCherry)20. The transgene was packed into a lentiviral vector. Two groups of

genetically engineered GCaMP6f mice were used in this experiment. Group one (n=4) was

injected with the above lentiviral vector at the somatosensory cortex, and group two (n=4) was


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injected with a lentiviral vector encoding mCherry without TRPV1 at the same brain location

(CaMKII-mCherry, Fig. 3a). After approximately 4 weeks for transgene expression, a cranial

window was created on top of the virus injection site and a glass window was added for 2PM

imaging of the neural activity by the genetically encoded calcium sensor GCaMP6f. A ring-

shape FUS transducer was coupled to the microscope objective through a custom-designed

adaptor for simultaneous FUS stimulation and 2PM imaging (Fig. 3a, 3b). The full-width-half-

maximum dimension of the FUS beam was 0.66 mm in the horizontal plane (XY plane, Fig. 3a),

which enables precise spatial control of the delivered FUS energy within the FOV of the 2PM

microscope.

We found a total of 13 neurons with co-expression of TRPV1 and GCaMP6f from mice

in group one. Two repeated FUS stimulations were delivered when these neurons were found in

the FOV. We observed that TRPV1+ neurons were switched from silent to active states in

response to FUS stimulation, while nearby neurons without TRPV1 overexpression were not

affected (Fig.3c). We consistently observed FUS activation of the TRPV1+ neurons, showing a

strong Ca2+ influx (Fig. 3e, right panel, and Fig. S5) and a high success rate of 80.8 ± 9.0% (Fig.

3f). For comparison purposes, we randomly selected 13 neurons from mice in group two with the

overexpression of mCherry without TRPV1 (TRPV1-). In these mice, FUS failed to activate

these selected neurons as seen from the minimal Ca2+ influx (Fig. 3e, left panel) and a low

success rate of 10.7 ± 5.7% (Fig. 3f), which was not significantly different from the control

without FUS stimulation (Fig. 3d, 3f). These findings demonstrated the ability of sonogenetics to

manipulate the activity of a selected type of neurons in the mammalian brain via TRPV1.

One practical consideration with thermal-based neuromodulation tools is the risk of

damaging effects from the temperature increase. Two additional groups of normal mice without


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the injection of the viral vectors were used to evaluate the safety of FUS exposure (n=4 for each

group). One group was sacrificed after FUS sonication with identical parameters as those used in

the above experiment (FUS+). The other group was used as the control without FUS exposure

(FUS-). We inspected neuronal integrity, inflammation, and apoptosis using

immunohistochemical staining of the neurons (NeuN), astrocytes (GFAP), microglia (Iba1), and

cell death (Caspase-3 and TUNEL). Cell nuclei appeared to be intact and the morphology of

neurons appeared normal. We did not find a significant change in the numbers of neurons,

astrocytes, microglia, and apoptotic cells when comparing FUS+ and FUS- groups.


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Fig 3. Sonogenetics in mouse brain in vivo. (a) Schematic illustration of the experiment

workflow. The FUS pressure distribution in the 2PM imaging plane as measured by a

hydrophone is shown on the right. (b) A photo of the 2PM setup which couples a ring-shaped


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FUS transducer with the microscope objective using a customized adapter. The mouse head was

fixed by a holder to minimize motion artifact in 2PM imaging. An acoustic absorber was placed

underneath the mouse head to decrease the reflection of the ultrasound pulses from the bottom of

the mouse head. (c) A representative 2PM image of the mouse cortex in vivo is shown on the left.

The image on the right highlights two neurons (1 and 2 in pink) expressing both TRPV1-

mCherry and GCaMP6f and two neurons (3 and 4 in blue) expressing GCaMP6f only. The Ca2+

fluorescence intensity changes over time of the 4 neurons are shown next to the 2PM image. A

total of 13 neurons were selected for mice with and without overexpression of TRPV1 and each

neuron was stimulated by two repeated FUS stimulations. (d) Average of the Ca2+ fluorescence

intensity curves for mice with and without overexpressing of TRPV1 before FUS exposure,

which served as the control for without FUS stimulation (FUS-). (e) Average of the Ca2+

fluorescence intensity curves for mice with and without overexpression of TRPV1 with FUS

stimulation (FUS+) Solid lines are the mean of the 26 stimulations and the shaded area indicates

the SEM. (f) Comparison of the success rate of these selected neurons with and without FUS

stimulation. The error bar represents SEM. (g) Evaluation of neuronal integrity, inflammation,

and apoptosis after FUS exposure in the FUS-targeted location using immunohistochemical

staining of the neurons (NeuN), astrocytes (GFAP), microglia (iba1), Caspase-3, and TUNEL. In

the fluorescence images, blue indicates the DAPI stained nuclei and the other pseudo colors

indicate different cell types. (h) Percentages of positive-stained cells to DAPI cells comparing

without and with FUS exposure within the FUS targeted location.


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Precise control of neural activity

Precise control of neural activity via sonogenetics depends on well-defined temporal and

spatial control of the ultrasound energy. All the above experiments were performed using a 15 s-

long FUS pulsed wave. After demonstrating that the 15 s-long FUS pulsed wave can robustly

activate TRPV1+ neurons (Fig. 3), we compared pulsed wave (PW) with continuous wave (CW)

for targeted activation of TRPV1+ neurons. The experimental method was identical to the above

in vivo study with one modification. Considering the relatively low co-expression rate of TRPV1

and GCaMP6f using the transgenic mice, we switched to co-injection of two viruses: one for the

delivery of GCaMP6f and one for the delivery of TRPV1.

Short on- and off-time delays of neuromodulation permit multiple simulations in short

observation times, which is crucial for precise correlation of neural activity with brain function

and for obtaining statistically significant data. We tested whether we could shorten the

stimulation duration by using PW FUS with 7 s total sonication duration and CW FUS with 7, 4,

and 1 s sonication duration. Each condition was tested on 5 TRPV1+ neurons and each neuron

was repeatedly stimulated 10 times. We found that reducing the PW duration to 7 s failed to

evoke Ca2+ influx robustly, but CW with 7 s and 4 s duration repeatedly activated TRPV1+

neurons in the mouse brain in vivo (Fig. 4a, Supplementary Video 1). Further decreasing the

CW duration to 1 s did not consistently induce activation of the neurons. The success rate of 7-s

CW and 4-s CW (88.0 ± 7.3% and 68.0 ± 11.6% respectively) were comparable to 15-s PW

stimulation (80.8 ± 9.0%) with no significant difference (Fig. 4b). The onset latencies were 2.6 ±

0.2 s and 2.1 ± 0.3 s for the 7-s CW and 4-s CW stimulation, respectively (Fig. 4c). They were

significantly faster than the latency for the 15-s PW (5.9 ± 1.0 s). In addition to faster onset time,

CW also reduced the 50% relaxation time. The normalized fluorescence intensity (ΔF/F) was


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back to half of its peak intensity within 14.6 ± 1.0 s and 15.3 ± 2.0 s for the 7 s and 4 s

stimulation, respectively (Fig. 4d).

We performed in vivo real-time temperature imaging using magnetic resonance (MR)

thermometry. MR thermometry is an established technique for noninvasive, real-time,

volumetric, and quantitative temperature measurements. The most reliable and commonly used

MR thermometry method is based on temperature-sensitive proton resonance frequency shift.

We have established the MR thermometry technique for monitoring heating by FUS in our

previous studies24,25. MR thermometry shows localized heating at the FUS spatially targeted

brain location. The small FUS beam size (Fig. 3a) allows us to precisely target at the cortex

underneath the optical window. We found that heat induced by FUS was spatially localized at the

targeted brain location (Fig. 4e). We quantified the average temperature change within the

optical window for both PW-FUS and CW-FUS stimulation (Fig. 4f). We then calculated the

peak temperature associated with each sonication condition and found that the success rate of

TRPV1+ neuron activation was positively correlated with the peak temperature (Fig. 4g). This

positive correlation between temperature and success rate further supports our hypothesis that

FUS-induced heating is the dominant mechanism for TRPV1-mediated sonogenetics. The

temperature curves also verified that CW FUS raised the temperature faster than PW FUS (Fig.

4f), which could explain the shortened latencies associated with 7-s CW and 4-s CW compared

with 15-s PW. These findings suggest that we can control the success rate, on-off temporal

dynamics, and spatial location of sonogenetic neuromodulation by using different FUS

parameters to effectively control the heating effect.


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Fig. 4. Precise control of neural activity by sonogenetics. (a) Illustration of different FUS

parameters for stimulation (Left). The representative Ca2+ dynamics in response to 10 repeated

FUS stimulations using different parameters (Right). Yellow bars indicate the FUS stimulation

time. PW: pulsed wave. CW: continuous wave. Quantification of (b) Success rate, (c) latency,

and (d) time to 50% relaxation for different FUS parameters (error bars indicate SEM). *:

P<0.05; **: P<0.002; ***: P<0.001. (e) Spatial distribution of FUS-induced heating as imaged

by MRI thermometry in vivo. The dashed circle indicates the location of the optical window. An

image of the mouse with the optical window is shown on the left. (f) Mean temperature within


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the optical window as a function of time for both PW and CW stimulation. The yellow solid line

indicates the onset of FUS stimulation. (g) Correlation between the peak temperature associated

with each FUS parameter and the success rate of TRPV1+ neuron activation. Error bars indicate

SEM.

Discussion

This study demonstrated that TRPV1-based sonogenetics is a noninvasive, safe, cell-type

specific, and spatiotemporally controllable neuromodulation tool. ThermoTRP channels are

cation channels of the Transient Receptor Potential family whose conductances change

dramatically with temperature18. Among the identified thermoTRPs, TRPV1 is the most suitable

one for the mammalian application, because its temperature sensitivity threshold is

approximately 42 ºC17. TRPV1 has been used by magnetothermal genetics to modulate neural

activity19,20 and control animal behavior26,27. Alternating magnetic fields were used to heat

superparamagnetic nanoparticles adhering to the neuronal membrane for the activation of

neurons heat-sensitized by expressing TRPV1. Magnetothermal genetics allows noninvasive

delivery of energy to control neural activity remotely, but major safety issues arise from the

exogenous superparamagnetic nanoparticles incorporated into the brain and poor spatial

resolution of the magnetic field. FUS can remotely warm up the brain tissue under precise spatial

and temporal control without the need of injecting nanoparticles to the brain. The strong positive

correlation between temperature and the success rate of TRPV1+ neuron activation (Fig. 4g) has

not been reported before by magnetothermal genetics. Our findings that the success rate and the

on-off temporal dynamics of TRPV1-based sonogenetics can be precisely controlled by FUS


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suggest that our proposed sonogenetics is a controllable and reliable neuromodulation tool. Past

research effort has been focused on developing sonogenetics based on mechanosensitive ion

channels. One most recent study showed that an engineered auditory-sensing protein, prestin,

successfully stimulated neurons in deep regions of mouse brains as evaluated by c-fos staining of

ex vivo brain slices16. However, FUS-evoked calcium influx in HEK293T cells expressing this

protein lasted for more than 100 s after a single FUS stimulation with no evidence to show the

calcium influx could be turned off. Such poor temporal response limits the application of this

protein as a sonogenetic tool and the lack of understanding of how this protein senses ultrasound

further hinders its utilization in neuromodulation. Our study opens new avenues for developing

sonogentics using thermosensitive ion channels. Future studies are needed to screen other

candidate thermo- and mechano-sensitive ion channels to further expand the toolbox for

sonogenetics.

Although ultrasound is well-known to be associated with both mechanical and thermal

effects, existing efforts on ultrasound neuromodulation focus on harnessing the mechanical

effects of ultrasound while avoiding the heating effects. Heating effects have been intentionally

avoided for the major concern of tissue destruction as the gap between normal body temperature

and the noxious, tissue-damaging temperature is narrow18. However, technological advancement

has made it possible to real-time monitor and control FUS-induced heating in the human brain

based on MR thermometry28. Moreover, MR investigation of FUS-induced brain tissue damage

suggested a safety thermal dose threshold of about 1 equivalent min at 43 ºC for the brain29. The

thermal doses associated with our FUS parameters were within the range of 0.11 – 0.35

equivalent min at 43 ºC, which were much lower than the above safety threshold. The safety of

the FUS stimulation was confirmed by our inspection of neuronal integrity, apoptosis, and


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inflammation markers. We do not rule out potential damage by other FUS parameters or chronic

effects occurring over a longer time. Further studies are needed to fully establish the safe

operation parameter space of the proposed sonogenetic technique.

The sonogenetic tool based on TRPV1 has a latency of several seconds. This time scale is

not limited by the intrinsic properties of the ion channel, as the mammalian TRPV1 responds

with a time constant of ~5 ms to an infrared-laser triggered temperature jump, comparable to

ligand- or voltage-gated ion channels30. This time scale reflects the kinetics of FUS tissue

heating to reach the targeted temperature for neuron activation. The latency is longer than

optogenetics which has a millisecond temporal resolution. Millisecond temporal resolution is

unnecessary for many applications, for example linking activation of specific neurons to

behavior outputs18. Moreover, the second-scale latency by the sonogenetics is comparable to the

response time of magnetothermal neural stimulation27 but much faster than chemical stimulation

using designer receptors exclusively activated by designer drugs (DREADDs)31.

Sonogenetics, like all other genetic-based neuromodulation techniques, requires gene

therapy for genetic targeting of specific cell types to express ultrasound-sensitive ion channels.

Gene therapy methods based on viral and other vectors have already provided promising results

in clinical trials, and new genome editing technologies have brought safe gene delivery a step

closer to clinical practice32. Recently, several preclinical studies have shown that FUS in

combination with microbubbles can transiently enhance the blood-brain barrier permeability for

the noninvasive and localized delivery of intravenously injected AAVs to the mouse brain for

optogenetics33 and chemogenetics34. Currently, clinical trials are evaluating the feasibility and

safety of the FUS technique for drug delivery to the brain35–37. FUS has the promise to achieve

localized and noninvasive delivery of the viral vectors encoding ultrasound-sensitive ion


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channels and activation of the expressed ion channels for fully noninvasive cell-type-specific

neuromodulation.

Sonogenetics can accelerate achieving the ultimate goal of neuroscience in

understanding intact neural circuits and enable potential clinical applications. Compared with the

existing FUS neuromodulation technique, which uses ultrasound alone to activate or inhibit brain

regions, sonogenetics can target a specific type of neurons in the chosen brain area. This

specificity allows sonogenetics to be used to study of causal relationships between neural activity

and a behavioral outcome in a temporally precise manner. Further, optogenetics often requires

surgery for the implantation of optical fibers to the brain, which is one of the major challenges in

the engineering of optogenetics systems for clinical use. FUS energy can be noninvasively

delivered to targeted brain locations. The design of the FUS transducer can be modified to suit

different applications. For example, the geometry of the transducer can be designed to generate a

focal zone that closely matches the volume of the targeted brain tissue. Moreover, instead of

using a single-element FUS transducer, a phased array transducer with multiple elements can be

used to form various focus patterns38 for multiple-location simultaneous stimulation. The phased

array transducer can also electronically steer the beam for sequential stimulation of multiple

brain locations. Devices for transcranial delivery of FUS energy for clinical thermal ablation

treatment of essential tremor has been approved by the U. S. Food and Drug Administration39.

We envision that the sonogenetic technique could be extended to large animals and even humans

owing to the deep penetration depth of FUS.

Not only will sonogenetic have a broad range of applications in basic and translational

neuroscience, its principle of using FUS for noninvasive, spatiotemporal control of biological

systems will also impact other fields in biological science and biomedical engineering. In the


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future, such a technique may even support new therapeutic modalities for humans, enabling the

control of cells not only in the brain but also in other parts of the body.

Methods

Plasmid and virus. pLenti-CaMKII-TRPV1-p2A-mCherry-WPRE was a gift from P. Anikeeva

(MIT). Lentiviral vector was produced according to the established protocol by Hope Center

Core at Washington University in St. Louis. Briefly, HEK293T cells were plated at 30–40%

confluence 24 h before transfection (70–80% confluence at the time of transfection). 10 g of the

lentiviral vector with the appropriate insert, 5.8 µg of pMDLg/pRRE, 3.1 µg of pCMV-G, and

2.5 µg of pRSV-rev were cotransfected into HEK293T cells using the calcium phosphate

precipitation method. The medium was replaced with fresh medium containing 6 mM sodium

butyrate at 6 h after transfection. Culture supernatant was collected 42 h after transfection. The

supernatant was passed through a 0.45 µm SFCA syringe filter (Corning) and ultracentrifuged

through a 5 ml 20% sucrose (in PBS) cushion in a polyallomer tube at 11000 rpm, 4 °C, for 4 h

with SW28 rotor (Beckman Coulter). The concentrated vector was stored at -80 °C until

use. The vector titers were determined by real-time PCR as previously described40. To make

pLenti-CaMKII-mCherry-WPRE, the mCherry fragment was ligated to pLenti-CaMKII-TRPV1-

p2A-mCherry-WPRE in which TRPV1-p2A-mCherry was removed by digestion with BamHI

and EcoRI (blunt end-treated).

In vitro cell culture experiments. The HEK293T cell line was a gift from X. W. Wang

(Washington University in St. Louis). HEK293T cells were grown in DMEM (4.5 g/L glucose),

supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/mL sodium pyruvate, 1% non-

essential amino acids, and 1% PenStrep. 4×105 HEK293T cells seeded in a 6-well plate 1-day

prior were transduced overnight with 2 μL lentivirus of pLenti-CaMKII-mCherry-WPRE or


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pLenti-CaMKII-TRPV1-p2A-mCherry-WPRE in the presence of 8 μg/ml polybrene. One week

later, pLenti-CaMKII-mCherry-WPRE and pLenti-CaMKII-TRPV1-p2A-mCherry-WPRE

transduced HEK293T cells were enriched for mCherry positive cells (>95%) using fluorescence-

activated cell sorting (FACS) at Siteman Flow Cytometry Core at Washington University in St.

Louis. Sorted transfected cells were expanded and used for Ca2+ imaging and FUS stimulation.

Fluo-4 AM (Thermo Fisher Scientific), a Ca2+ indicator, was used to image the spatial

dynamics of Ca2+ response to FUS stimulation using a fluorescence microscope (LX70,

Olympus). First, a 50 μg Fluo-4 stock solution was diluted by 45 μL Pluronic F-127 20%

solution in DMSO (P3000MP, Thermo Fisher Scientific) and added to the HEK293T cells in a

48-well plate to the final concentration of 4 μM. After 45-min incubation at 37 ºC, cells were

washed in indicator-free live cell imaging solution (Thermo Fisher Scientific) twice and

incubated for another 30 min for complete de-esterification. Then the cells were immersed in the

live cell imaging solution and the Ca2+ images were recorded by the fluorescence microscope at

a frame rate of 2.4 frames/sec.

FUS stimulation was applied to the cells during Ca2+ imaging using a customized in vitro

FUS system (Supplementary Fig. S1). The ring-shaped transducer was aligned confocally with

the objective of the fluorescence microscope. Above the ring transducer were a layer of 0.2 mm

transparent plastic window and a thin layer of degassed ultrasound coupling gel. The wells were

filled with the live-cell imaging solution and covered by ultrasound absorber on top (Aptflex

F28P, Precision Acoustics) to minimize standing waves. The acoustic pressure output of the FUS

transducer was carefully calibrated using hydrophones (HGL-200 and HNP-200, Onda) in a

degassed water tank. The FUS parameters applied in the study were: center frequency of 1.7

MHz, peak negative pressure of 1 MPa, duty cycle of 40%, pulse repetition frequency of 10 Hz,


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and duration of 30 s. The spatial peak temporal average intensity (Ispta) was calculated to be 12.5

W/cm2. The temperature of the imaging solution was monitored using a fiber-optic thermometer

(Luxtron, now LumaSense Technologies). The tip of the fiber-optic thermometer was inserted at

a location about 1 mm away from the center of the microscope’s FOV for real-time temperature

recording. During the experiment, the baseline temperature of the medium was maintained at 37

ºC by a resistor-based heating unit (Caddock File). For the positive control experiment, the cells

were heated by water-bath heating using the resistor-based heating unit.

FITC Annexin V (AV) and propidium iodide (PI) were used to assess the effect of FUS

stimulation on cell viability. TRPV1- and TRPV1+ HEK293T cells were seeded in a 48-well

plate and treated by FUS. At 12 h, 24 h, and 48 h after sonication, cells were harvested and

stained with AV and PI according to manufacturer’s manual (FITC Annexin V Apoptosis

Detection Kit with PI; Biolegend). Stained cells were immediately analyzed by flow cytometry

(MACSQuant, Miltenyi Biotec) and the data were analyzed using Flowjo 8.7 software (Tree Star

Inc., Ashland) to quantify the percentages of necrotic cells (AV-, PI+), early apoptotic cells

(AV+, PI-), late apoptotic cells (AV+, PI+), and live cells (AV-, PI-).

Intracranial injection. Thy1-GCaMP6f mice (male, 8-week old, Jackson Laboratory) and wild-

type BALB/c mice (female, 8-week old, Jackson Laboratory) were subjected to intracranial

injection of viral vectors. All animal studies were performed in compliance with guidelines set

forth by the NIH Office of Laboratory Animal Welfare and approved by the Washington

University Institutional Animal Care and Use Committee. All mouse surgeries were performed

under aseptic conditions. Mice were anesthetized via intramuscular injection of ketamine (0.10

mg/g body weight) and xylazine (0.01 mg/g body weight) mixture. Before the virus injection,

buprenorphine (buprenex, 0.1 μg/g body weight) and carprofen (Rimadyl, 5 μg/g body weight)


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were injected subcutaneously. Coordinates used for intracranial injection into the primary

somatosensory cortex were determined according to the mouse brain Atlas as follows: -0.5 mm

dorsoventral, -1.2 mm anterior-posterior, and -1.2 mm mediolateral. Equivalent dose of lentivirus

(1.0 μL of pLenti-CaMKII-TRPV1-p2A-mCherry-WPRE solution or 0.64 μL pLenti-CaMKII-

mCherry-WPRE) was injected into the somatosensory cortex of Thy1-GCaMP6f mice using a

microinjector (Nanoject II; Drummond Scientific) at a speed of 0.69 μL /min. After the injection,

the needle was slowly withdrawn at a speed about 0.5 mm/min. The scalp was closed with

vetbond (3M) and sutured, and the mice were allowed to recover on a heating pad. Following the

surgery, anti-inflammatory and antibiotic drugs, including sulfamethoxazole (1 mg/ml),

trimethoprim (0.2 mg/ml), and carprofen (0.1 mg/ml), were chronically administered in the

drinking water. Following the same procedures, BALB/c mice were injected with a mixture of

1.0 μL Lentivirus (pLenti-CaMKII-TRPV1-p2A-mCherry-WPRE) and 0.1 μL of adeno-

associated virus serotype 9 (AAV9) carrying GCaMP6f under the excitatory neuronal promoter

CaMKII (AAV-CaMKII-GCaMP6f) (Addgene) to co-transfect the TRPV1 and GCaMP6f into

neurons.

In vivo 2PM imaging and FUS brain stimulation. 4–6 weeks following viral injection, mice

were used for FUS stimulation with simultaneous in vivo 2PM imaging to record the neural

activity. Before the experiment, a chronic cranial window was created on the mouse head to

obtain optical access to the mouse cerebral cortex for time-lapse Ca2+ imaging using 2PM

following an established protocol41. Briefly, a small piece of the skull (a circular shape with ~2.3

mm diameter) was removed by a trephine drill (Fine Science Tools). Then 1.2% agarose (Sigma)

in artificial cerebrospinal fluid (aCSF, Tocris Bioscience) was applied onto the dura and covered

by a 3.5 mm cover glass (#1 thickness, CS-3.5R, Warner instruments). The cover glass was then


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24

sealed by vetbond and adhesive cement (C&B-Metabond). Finally, a customized titanium frame

was embedded on top of the skull and fixed by adhesive cement. After the optical window

surgery, the mice were prepared for 2PM imaging and FUS stimulation. The mice were

anesthetized via intramuscular injection of ketamine (0.10 mg/g body weight) and xylazine (0.01

mg/g body weight) mixture. Then, the head was fixed by screwing the titanium frame on top of

the mouse’s head to a titanium plate on the 2PM stage (Fig. 3b). Body temperature was kept

around normal physiological status (37 °C) by an automatic temperature controller (TC-334B,

Warner Instruments).

Time-lapse 2PM imaging was performed with a custom-built two-photon microscope42

running SlideBook acquisition software (Intelligent Imaging Innovations) and equipped with a

Thor resonant scan head, 2 Bialkali and 2 multi-alkali photomultiplier tubes (Hamamatsu), a 1.0

NA 20× water dipping objective (Olympus), and a Pockel's cell laser attenuator (Conoptics).

Fluorescence was excited with a Chameleon Vision II Ti: Sapphire laser (Coherent) tuned to 920

nm for GCaMP6f and 980 nm for mCherry; fluorescence emission was detected by

photomultiplier tubes simultaneously using 495 nm and 575 nm dichroic filters: green (495–575

nm, GCaMP6f) and red (>575 nm, mCherry). The output laser power was set to around 60 mW

for Thy1-GCaMP6f mice and 20 mW for AAV-CaMKII-GCaMP6f transfected mice. The image

resolution was 0.585 µm/pixel. The original scanning frame rate was 30 Hz and every 10 frames

were averaged to generate time-lapse images with 3 Hz frame rate.

The FUS transducer was specially designed in the way that the inner edge of the ring

FUS transducer geometrically fit the outer edge of the microscope objective to confocally align

the optical beam and FUS beam. In the repeated FUS stimulation studies, the interval between

every adjacent stimulation was 80 s to minimize interference among repeated stimulations. A


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25

total of 5 different parameter groups were used in the in vivo study with the ultrasound frequency

(1.7 MHz) and peak negative pressure (1.3 MPa) kept the same and changing the duty cycle and

sonication duration: (1) PW with duty cycle of 40% and duration of 15 s; (2) PW with duty cycle

of 40% and duration of 7 s; (3) CW with duty cycle of 100% and duration of 7 s; (4) CW with

duty cycle of 100% and duration of 4 s; (5) CW with duty cycle of 100% and duration of 1 s;

Calcium data analysis. The recorded Ca2+ images from both in vitro cell culture and in vivo

mice studies were analyzed by Matlab using a published algorithm43. For the in vitro experiment,

cells were automatically identified after applying a constrained nonnegative matrix factorization

(CNMF) framework. Then, 100 cells are randomly selected from 3 independent trials. Relative

fluorescence intensity changes were computed for calcium signal as ΔF/F=(F-F0)/F0, where F0

represents the average of a 1.5 s-long fluorescent signal acquired before FUS onset. Successful

FUS stimulation was defined by the criteria that the normalized Ca2+ fluorescence intensity

(ΔF/F) acquired from the onset of FUS stimulation to 1.5 s after FUS was both >0.1 and > 2×

standard deviation (SD) of 1.5 s-long signal acquired before FUS44. The percentage of

responsive cells was calculated by dividing successfully stimulated cells over all the selected

cells.

For the in vivo study, ROIs were manually selected to cover individual soma of the

neurons expressing both GCaMP6f and mCherry with TRPV1 for TRPV1+ neurons and without

TRPV1 for TRPV1- neurons. Successful FUS stimulation was defined as the above in vitro study

(ΔF/F > 0.1 and >2× SD). The success rate was quantified by the proportion of successful FUS

stimulation to all the stimulation in every single neuron. The statistical result was then calculated

by averaging the success rate over all mCherry and GCaMP6f double-positive neurons. Latency

to threshold was defined as the time from the onset of FUS to the time of successful stimulation,


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26

which was defined above. Time to 50% relaxation was defined as the time from the time

reaching the peak amplitude to the time decaying to half of the peak amplitude.

In vivo temperature imaging. MR thermometry was used for non-invasively imaging the

spatiotemporal distribution of FUS-induced temperature rise in mice brain in vivo. BALB/c mice

without viral injection were used in this study. MR thermometry was performed using a

(Agilent/Varian DirectDrive Console) 4.7 T MRI system. Temperature images were acquired

using a continuously applied gradient-echo imaging sequence with a flip angle of 20 degrees,

TR of 10 ms and TE of 4 ms, slice thickness of 1.5 mm, and matrix size of 128 × 128 for 60 × 60

mm FOV. Phase images were processed in real-time using ThermoGuide software (Image

Guided Therapy). MRI compatible FUS transducer (Image Guided Therapy) was targeted at the

same brain location as 2PM study and used the same FUS parameters as well, except a slight

difference in the ultrasound frequency (1.5 MHz instead of 1.7 MHz), which was restricted by

the available transducer. Before the in vivo experiment, the performance of the MR thermometry

was calibrated in a gelatin phantom by comparing it to the temperature measured by the fiber-

optic thermometer used as a gold standard. The difference in the result of these two methods was

within 10%.

Histological analysis. Safety of FUS stimulation was assessed by evaluating the number of

neurons, astrocytes, microglia and apoptotic cells using immunohistochemistry analysis. First, 5

sequential FUS stimuli were applied to the BALB/c mice without viral injection using the same

FUS system as the above MR thermometry study and the same FUS parameters. At around 2 h

after FUS sonication, mice were sacrificed by transcardial perfusion with 4% paraformaldehyde

(PFA). Brains were harvested and fixed in 4% PFA overnight and equilibrated in 30% sucrose

for cryosectioning. The fixed brains were sectioned to 20 µm slices. Then, the slices were


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27

preprocessed in 0.3% v/v Triton X-100 and 3% v/v blocking serum solution in PBS for 1 h in the

dark at room temperature to increase the permeability and block the background. Then the slices

were washed 3 times using PBS and incubated in primary antibody solution with 3% blocking

serum overnight at 4 °C. The primary antibodies used in this study include: anti-NeuN (Abcam,

Cat: 104225, 1:1000), anti-GFAP (Abcam, Cat: 07165, 1:1000), anti-Iba1 (Abcam, Cat: 178846,

1:1000,), anti-Caspase-3 (Cell Signal, Cat: 9661s, 1:1000), and DeadEnd™ colorimetric TUNEL

System (Promega, Cat: G7360). After 3 PBS wash, the slices were then incubated in secondary

antibody (Donkey antirabbit Alexa Fluor 488, Jackson laboratory, 1:400) in the dark for 3 h at

room temperature. Finally, the slices were moved onto glass slides and mounted with

VECTASHIELD (Vector Laboratories) that contained DAPI nuclear stain.

Statistical analysis. Data were analyzed using either a two-tailed t-test with unequal variance (in

Fig. 3h) or two-way ANOVA with Bonferroni post hoc test (when more than two samples were

compared in Fig 2d, 3f). One-way ANOVA with Bonferroni post hoc test was used in Fig 4b, 4c,

and 4d. All data with p < 0.05 were considered to be significant.

Data availability. The data that support the findings of this study are available from the

corresponding authors upon reasonable request.


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28

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Acknowledgments

This work was supported by the National Institutes of Health (NIH) BRAIN Initiative

(R01MH116981) and NIBIB (R01EB027223). We thank Dr. Hunter Banks, Dr. Lu Zhoa, and

Ms. Lindsey Brier for the insightful discussions. We also thank D. Mingjie Li from the Hope

Center Viral Vectors Core at WashU for preparing the lentiviral and AAV vectors. We also

appreciate the help of Dr. James D. Quirk in thermometry mapping using magnetic resonance

imaging.

Author contributions

H.C. and Y. Y. designed the experiments. Y. Y. conducted the experiment with the assistant of

other co-authors. J.Y. assisted with the in vitro experiment. D.Y and M.M assisted with setting

up the two-photon microscopy system. L.Z and C.P assisted with the MR thermometry

experiment. H.B and Y.Y assisted with animal surgery. Y.Y performed the

immunohistochemistry staining experiment. J.C, J. P.C, and M. R.B contributed to the discussion

and interpretation of the results. H.C and Y.Y wrote the manuscript and all authors modified and

revised the manuscript.

Competing interests

The authors have declared that no competing interest exists.


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